PCR-based nucleic acid detection typically employs two primers that anneal to opposite strands of a DNA target. PCR can be multiplexed to amplify several targets in a well or droplet. One of the challenges of PCR is that primers can prime non-specifically on themselves forming primer dimers or on DNA targets that are similar to intended targets. This non-specific priming can occur during single-plex PCR, but is markedly more likely in multiplex PCR. For example, 10-plex PCR requires 10 primer pairs (20 primers) that can form 19+18+ . . . 1=19*10=190 different pairs, each potentially causing a non-specific amplification product such as a primer dimer.
Various methods have been described for overcoming non-specific PCR amplification caused by non-specific priming. The U.S. Pat. No. 5,792,607 (Backman et al) describes a method using EndoIV to unblock primers for PCR and ligation reaction. But because the activation of primers using EndoIV is very slow (minutes instead of seconds), it is impractical to use this method to amplify DNA exponentially. Also, the Backman patent only describes EndoIV derived from E. coli and only mentions that the method should work better if using thermostable EndoIV. The method described by Backman et al. has not been commercialized.
Recently IDT (Integrated DNA Technologies) published methods and made reagents available for RNAse H-dependent PCR (rhPCR, patent application US 2009/0325169; Dobosy et al., BMC Biotechnology 2011, 11:80). The authors describe a method using primers with blocked 3′-ends and containing a single RNA base close to the 3′-end. Primer activation occurs if a thermostable RNAseH enzyme cleaves the annealed primer at the RNA base, generating a 3′-OH, thereby making the primer extendable by polymerase. The rhPCR makes PCR more complex as it requires an additional thermostable enzyme, RNAse H, that is currently commercially available only from IDT and Epicentre. Also, an RNA base in the primer requires a more complex manufacturing and can double the cost of primer compared to a regular DNA oligo. For example, a list price per base at the 100 nmolar scale is $0.55 and $6.50 per base for DNA and RNA bases, respectively (IDT Technologies).
Next generation sequencing (NGS) often requires enrichment of regions of interest in the genome or transcriptome. PCR, single-plex or multiplex, is one of the methods frequently used for enrichment, for example, AmpliSeq from Life Technologies. There are three major challenges in multiplex PCR enrichment for NGS: 1) formation of primer-dimers; 2) carry over contamination; and 3) PCR-errors.                1. Primer Dimers: Primer dimers can consume a lot of reagents as short primer dimer amplicons tend to be amplified very efficiently and dominate the PCR reaction.        2. Carry over Contamination: The high cost and sequencing throughput of NGS, where millions of sequences are generated in a single run, results frequently in multiple samples with different bar-codes being combined and processed together on the same plate or strip. Opening reaction tubes after PCR can result in a few amplicons being transferred through aerosols into other reaction tubes; this is called carry over contamination.        3. PCR Errors: Polymerases tend to make errors during PCR (most frequently mis-incorporation of nucleotides) and, if these errors occur during early cycles they appear as “mutations” in NGS. Molecular bar-codes called degenerate base regions (DBR; see J. Casbon, S. Brenner et. al “Increasing confidence of allele calls with molecular counting,” U.S. Pat. No. 8,481,292, and U.S. patent application Ser. No. 13/853,981 “Method for accurately counting starting molecules”) are random sequence tags that are attached to molecules that are present in the sample. These tags allow one to distinguish PCR errors during sample preparation from mutations that were originally present in the sample.        
Previously Bi and Stambrook, [“Detection of known mutation by proof-reading PCR”; Bi and Stambrook, Nucleic Acids Research, 1998, Vol. 26, No. 12 3073-3075] and Lin-Ling et al. [“Single-base discrimination mediated by proofreading inert allele specific primers”; Lin-Ling et al., J Biochem Mol Biol. 2005 Jan. 31; 38(1):24-7] described how exo+ polymerases remove mismatched bases at the 3′-end. They specifically teach this as a genotyping or mutation detection method. Mutations are amplified by wild type specific primers that have a non-Watson-Crick base pair at the 3′ ends that are cleaved and extended; primers that perfectly match wild type are not cleaved and thus not extended. Similarly, PCT/US2005/010782 “Quantitative amplification with a labeled probe and 3′ to 5′ exonuclease activity” by Bin Li et. al. specifically teaches that the 3′ most “N residue represents a mismatch to the target nucleic acid sequence”. Also, patent EP 2324124 “Proofreading primer extension” by Fiss and Myers teaches that extension during PCR happens “if the 3′ portion of the oligonucleotide is not 100% complementary to the template nucleic acid”. The “proof-reading PCR” is rarely used in practice as seen by its low citations number: 36 citations for Bi and Stambrook and 5 for Lin-Ling (Google Scholar, January 2014).
The idea of combining tagged target specific and universal primers is known; see, for example, application PCT/US2010/029854 by May et al. “Multi-primer amplification method for bar-coding of target nucleic acids”. The “four primer amplicon tagging” method developed by Fluidigm uses closed tube amplification to incorporate sample bar codes for next generation sequencing (NGS) enrichment. But the method works only for singleplex PCR: each well has a pair of target-specific primers and a pair of universal, thus the name “four primer”. Fluidigm also offers 10-plex PCR enrichment, but it is an open tube protocol: bar coded primers are added after multiplex PCR. Recently, similar methods were published for NGS enrichment using 60-plex 4 nM 5′-tagged primers for 6 PCR cycles and then 2 μM universal primers are added (Nguyen-Dumont et, “A high-plex PCR approach for massively parallel sequencing,” Biotechniques, 55, pp 69-74, 2013). Nguyen-Dumont et al. also review different methods for NGS enrichment.
The primer concentrations are 50 to 1,000× lower than the 50 nM-1 μM used in traditional PCR. Low primer concentrations decrease the chance of primer dimer formation proportionally to the square of primer concentrations. However, concentrations of 5 nM and below generally do not generate a sufficient amount of PCR products to observe a signal in qPCR or a band on a gel.
Due to the above-noted drawbacks of current strategies to prevent non-specific amplification during PCR, e.g., specificity, cost, manufacturing logistics, and the like, there is a need for more specific, flexible, and cost-effective methods of PCR nucleic acid detection. The present invention meets these and a variety of other needs.